Histology

Cheng Hong: Histology Laboratory

Receiving specimens

1) The specimens will be sent down from the operating theaters/ clinics to the lab by a porter.
2) Upon receiving the specimens the Medical Technologist(MT) must check the total number of bags sent to the lab and check the patient’s particulars and the type of specimen with the request form.
3) Initial on the receiving form and stamp the time.
4) Sign the specimen log book state the number of bags received and the time.
5) Sort out the specimens according to: Placenta/ Point Of Conception(POC)/ Specimens pending for trimming/ Others.
6) The Laboratory Assistant(LA) will then give the specimens a lab assertion number.


Tissue Processing

Specimens for trimming
1) For placenta specimen/ specimens that needs trimming the Pathologist Assistance(PA) will do the trimming and will determine the number of tissue cassettes to use.
2) The PA will dictate the measurements and the characteristics of the specimens to the LA to record
3) The cassettes are then placed in the tissue processing rack and immerse it in buffered formalin.

Product Of Conception
1) POC are stored in sealed packets(to prevent spills/leakages to protect the MT’s safety)
2) POC is processed by the MT: the POC bag is cut into half and the MT must identify the chronic villi and place into cassette.
3) The MT must record the amount of sample used, number of cassette used and if there is any reserve. (Also must indicate if there is any fetal part seen)
4) The cassettes are placed in the tissue processing rack and immerse it in buffered formalin.

Tissue Processor
1) The cassettes in the holder is then placed into the Automated Tissue Processor(Shandon Excelcior).
2) We are able to program the tissue processor to start at a specific time and we are able to choose the different programs(eg:overnight/ rapid etc)
3) The steps are processed as shown below:

1) 10% formalin 30mins(To fix the tissue & preserve cells & tissue constituents)
2) 10% formalin 30mins
3) 70% alcohol 30mins (To remove fixative and water from tissue& replace them with alcohol)
4) 95% alcohol 30mins
5) Absolute alcohol 1hr
6) Absolute alcohol 2hrs
7) Absolute alcohol 2hrs
8) Xylene 30mins (Clearing: Replacing dehydrating fluid with fluid that is miscible with dehydrating fluid & embedding medium)
9) Xylene 30mins
10) Xylene 30mins
11) Paraffin 2.5hrs (wax replacing clearing agent with embedding medium)
12) Paraffin wax 3hrs

*We do not put the tissue into 100% alcohol directly as it will distort the tissue(must be a graded process)

Tissue Embedding
1) After the tissues have been processed, remove the holders and check with the log sheet if the assertion number tallies.
2) Open the cassette carefully and depending on the tissue size, choose a suitable mould size that will fit the tissue so that the entire surface will be exposed when obtaining the sections.
3) Dispense some wax into the mould and place the tissue into the mould and place on the cold plate and press the tissue evenly using a forceps or a pressing block( to ensure all surface will be exposed when sectioning).
4) Place the bottom portion of the cassette on top of the mould and dispense more wax until it fills almost half of the cassette.
5) Allow the wax to cool down for awhile before placing on the ice block(to speed up the hardening of the wax)
*It is important to embed the tissue in the correct orientation so that when the pathologist view the sections it will be in the correct orientation.

*It is also important to be quick when embedding, if the wax hardens before the the tissue is pressed out evenly, problems will arise when obtaining sections.

*For small biopsy, it is important to use a very hot mould so that we can expose the tissue as much as possible to be able to obtain good sections.


Shaving
1) After embedding, the blocks must be shaved first before cutting
2) The MT must be able to know how much to shave and to expose the tissue to prevent shaving off too much of the tissue thus not enough tissue is enough to obtain sections
3) For blocks that have blood clots we can soak them in water for about 5mins to soften the clot for easier cutting
4) For blocks that are fibrous(eg:cervix) we can soak them in softener(commercial softener/ soflant)
5) For blocks that are calcified we can do a surface decalcification by placing it in RDO
6) After steps 2 to 5 we place the blocks on the cold plate before cutting


Paraffin Sectioning
1) The aim is to obtain 3micron thick sections without folds on the tissue.
2) The blocks must be cold to obtain thin sections.
3) Insert the blade and check the thickness setting.
4) Place the block into the tissue block holder and adjust the distance from the blade using the coarse adjustment.
5) Turn the rotary knob away from you to obtain sections.
6) Slowly obtain ribbons and place on water and do any necessary adjustments(removing overlaps/folds).
7) Pick desired section and prepare a clean glass slide and fish up the section.
8) Place the section into a warm water bath(the section will spread out) and fish it up.
9) Let the slide stand for awhile before placing it in the slide holder.
10) Place the rack containing the slides into the oven set at around 82oC for 15mins(this is to let the tissue to “stick to the slide” to prevent it from floating off when staining).

*For sections that need to be cut in levels to slides must be obtain


Routine Haematoxylin and Eosin stain

1) Place the whole slide holder into the Lecia Autostainer and select the respective program
2) The steps as follows below:

(a) Dewax section in xylene for 5 minutes.
(b) Place in another xylene for another 5 minutes.
(c) Rehydrate section with absolute alcohol for 2 minutes.
(d) Place in another absolute alcohol for another 2 minutes.
(e) After then, place in 95% alcohol and 70% alcohol for 1 minute each.
(f) Rinse section with water.
(g) Stain the section with Harris Haematoxylin for 5 minutes.
(h) Rinse section with water.
(i) Differentiate staining with acid alcohol for 5-20 seconds.
(j) Blue the haematoxylin stain with running water or alkaline water.
(k) Check the differentiation microscopically.
(l) Stain the section with eosin for 1 min.
(m) Dehydrate section in graded alcohols with 70% alcohol (10 dips), 90% (10 dips) and 2 absolute alcohols for 3 min each.
(n) Clear the section in xylene for 5 min each with 2 changes.

3) After staining place the slide holder into the auto coverslipper.

*The haematoxylin will stain the nuclei blue or black and eosin will stain the cytoplasm red or pink.

*Eosin is the most suitable dye to combine with alum haematoxylin. It has the ability to distinguish between the cytoplasm of different types of cells and can also distinguish different types of connective tissue fibers.

*Stain results:
Nuclei: blue
Cytoplasm: varying shades of pink
Muscle fibers: deep pinky red
Red Blood cells: orange red
Collagen: pale pinky red
Fibrin: deep red


Frozen Sectioning

1) The lab will receive a call from the Operating Theater(OT) stating the OT number and type of specimen
2) A MT will be sent up to the OT to collect the sample immediately and the pathologist will be informed
3) Upon receiving the specimen, ensure that the patient ID tallies and record the time received and immediately sent back to the lab.
4) Prepare the equipments and tools needed for the pathologist and inform them when it is ready
5) The pathologist will dictate the characteristics and measurements of the specimen and mark out the orientation of the specimens using different colour dyes.
6) About 1 to 2 samples will be cut from the specimen into size about 15mm length and with and about 3to4mm thick.
7) Squeeze some freezing medium on to the tissue holder and place the tissue on it and immerse it into liquid nitrogen for about 5to8 seconds.
8) Place the tissue holder with the frozen tissue into the freezing microtome and shave off some tissue to obtain a full face.
9) Cut the tissue and using a glass slide to pick up the section( the temperature difference of the tissue(-35oC) and the glass slide(RT) will cause the freezing medium to melt and adhere to the slide.
10) Proceed to Rapid Staining


Rapid Staining
1) Xylene 10dips
2) Wash in running water
3) Haematoxylin 1min
4) Wash in running water until no more haematoxylin runs off
5) Ammonia 3 to 5 dips
6) Wash in running water
7) Eosin 10dips
8) 70% alcohol 10dips
9) 95% alcohol 10dips
10) 100% alcohol 10dips
11) Xylene 10dips
12) Xylene 10dips
13) Mount with DEPEX
14) Slide viewed by pathologist and the pathologist will call the doctor in the OT about the diagnosis.

*The whole process must be done within 20mins as the patient in the OT is still unstitched, the doctor must know if they have removed all the tumor from the body.